Evaluation of recombinant adenovirus-mediated gene delivery for expression of tracer genes in catecholaminergic neurons

Selective labeling of small populations of neurons of a given phenotype for conventional neuronal tracing is difficult because tracers can be taken up by all neurons at the injection site, resulting in nonspecific labeling of unrelated pathways. To overcome these problems, genetic approaches have been developed that introduce tracer proteins as transgenes under the control of cell-type-specific promoter elements for visualization of specific neuronal pathways. The aim of this study was to explore the use of tracer gene expression for neuroanatomical tracing to chart the complex interconnections of the central nervous system. Genetic tracing methods allow for expression of tracer molecules using cell-type-specific promoters to facilitate neuronal tracing. In this study, the rat tyrosine hydroxylase (TH) promoter and an adenoviral delivery system were used to express tracers specifically in dopaminergic and noradrenergic neurons. Region-specific expression of the transgenes was then analyzed. Initially, we characterized cell-type-specific expression of GFP or RFP in cultured cell lines. We then injected an adenovirus carrying the tracer transgene into several brain regions using a stereotaxic apparatus. Three days after injection, strong GFP expression was observed in the injected site of the brain. RFP and WGA were expressed in a cell-type-specific manner in the cerebellum, locus coeruleus, and ventral tegmental regions. Our results demonstrate that selective tracing of catecholaminergic neuronal circuits is possible in the rat brain using the TH promoter and adenoviral expression

Inference for stochastic chemical kinetics using moment equations and system size expansion

Quantitative mechanistic models are valuable tools for disentangling biochemical pathways and for achieving a comprehensive understanding of biological systems. However, to be quantitative the parameters of these models have to be estimated from experimental data. In the presence of significant stochastic fluctuations this is a challenging task as stochastic simulations are usually too time-consuming and a macroscopic description using reaction rate equations (RREs) is no longer accurate. In this manuscript, we therefore consider moment-closure approximation (MA) and the system size expansion (SSE), which approximate the statistical moments of stochastic processes and tend to be more precise than macroscopic descriptions. We introduce gradient-based parameter optimization methods and uncertainty analysis methods for MA and SSE. Efficiency and reliability of the methods are assessed using simulation examples as well as by an application to data for Epo-induced JAK/STAT signaling. The application revealed that even if merely population-average data are available, MA and SSE improve parameter identifiability in comparison to RRE. Furthermore, the simulation examples revealed that the resulting estimates are more reliable for an intermediate volume regime. In this regime the estimation error is reduced and we propose methods to determine the regime boundaries. These results illustrate that inference using MA and SSE is feasible and possesses a high sensitivity

Development of 18^{18}F-labelled radioligands for molecular imaging of the dopamine D4_{4} receptor

The five subtypes of the dopamine receptor play an important role in the human brain. The dopamine D$_{4}$ receptor is involved in processes of behaviour control and is assumed to be responsible for the emergence of the attention deficit hyperactivity disorder (ADHD) as well as psychotic diseases like schizophrenia. While most of the other dopamine receptors are well known there is a lack of suitable radioligands for the examination of the D$_{4}$ receptor by functional neuroimaging via positron emission tomography (PET). This is due to the extremely low distribution density of D$_{4}$ in the central nervous system. In this work the radiosynthesis of such D$_{4}$ ligands was developed and pharmacologically evaluated. Therefore, selected pharmaceutical lead structures were labelled via nucleophilic substitution with no-carrier-added (n.c.a.) [$^{18}$F]fluoride at an aromatic ring and subsequently coupled in a 1-2 step build-up reaction to the desired ligands. As first approach, an efficient radiosynthesis of the highly selective [$^{18}$F]FAUC 316 ligand ([$^{18}$F]1) was developed. Starting from $^{18}$F-labelling of the symmetric iodonium salts bis(4-bromophenyl)iodonium triflate and bis(4-iodophenyl)iodonium triflate the corresponding 4-[$^{18}$F]fluorohalobenzenes were obtained in radiochemical yields (RCY) of up to 60 %. Pd-catalyzed cross-coupling of the labelling products and piperazine with Pd$_{2}$(dba)$_{3}$ or Pd(OAc)$_{2}$ led to 4-[$^{18}$F]fluorophenylpiperazine in a RCY of up to 42 %. During the synthesis of standard and precursors 5-Cyanoindol-2-carbaldehyd was synthesized in four reaction steps with an overall yield of 15 % and coupled to [$^{18}$F]FAUC 316. The overall-RCY after high performance liquid chromatography (HPLC) separation was 10 %. [$^{18}$F]FAUC 316 was not suitable for further evaluation steps in vivo due to the very high nonspecific binding content determined by in vitro autoradiography. Alternatively, the radioligands 6-(4-[4-$^{18}$F]fluorobenzyl]piperazine-1-yl)benzodioxine ([$^{18}$F]$\textbf{33a}$), 6-(4-[4-[$^{18}$F]fluoro-(3-methoxybenzyl)] piperazine-1-yl)benzodioxine ([$^{18}$F]$\textbf{33b}$), 6-(4-[4-[$^{18}$F]fluoro-(3-hydroxybenzyl)]piperazine-1-yl)benzodioxine ([$^{18}$F]$\textbf{33d}$) und 6-(4-[6-[$^{18}$F]fluoropyridine-3-yl]piperazine-1-yl)benzodioxine ([$^{18}$F]$\textbf{33e}$) were synthesized as benzodioxine derivatives with decreasing lipophilicity. For this 1-(1,4-benzodioxine-6-yl)piperazine ($\textbf{30}$) was coupled with the corresponding aldehyde derivatives by a reductive amination reaction in overall-RCY of 35 %, 20 %, 9 % and 15 %, respectively. $\textit{In vitro}$ autoradiography on rat brain slices confirmed the correlation between non-specific binding and lipophilicity and lend [$^{18F}]\textbf{33d}$ and [$^{18F}]\textbf{33e}$ as putative radiotracers. Since [$^{18}F]\textbf{33e}$ showed better D$_{4}$ selectivity, $\textit{ex vivo}$ organ uptake, metabolization rate and brain distribution were determined. Examinations showed a principle qualification of [$^{18}F]\textit{33e}$ for the visualization of the D$_{4}$ receptors, but due to a lack of experiences a clear relation of D$_{4}$ to the ligand was not possible up to now. Further examinations in vivo are required to verify the ability of mapping D$_{4}$ receptors by this new radioligand

4-[18F]Fluorophenylpiperazines by Improved Hartwig-Buchwald N-Arylation of 4-[18F]fluoroiodobenzene, Formed via Hypervalent λ3-Iodane Precursors: Application to Build-Up of the Dopamine D4 Ligand [18F]FAUC 316

Substituted phenylpiperazines are often neuropharmacologically active compounds and in many cases are essential pharmacophores of neuroligands for different receptors such as D2-like dopaminergic, serotoninergic and other receptors. Nucleophilic, no-carrier-added (n.c.a.) 18F-labelling of these ligands in an aromatic position is desirable for studying receptors with in vivo molecular imaging. 1-(4-[18F]Fluorophenyl)piperazine was synthesized in two reaction steps starting by 18F-labelling of a iodobenzene-iodonium precursor, followed by Pd-catalyzed N-arylation of the intermediate 4-[18F]fluoro-iodobenzene. Different palladium catalysts and solvents were tested with particular attention to the polar solvents dimethylformamide (DMF) and dimethylsulfoxide (DMSO). Weak inorganic bases like potassium phosphate or cesium carbonate seem to be essential for the arylation step and lead to conversation rates above 70% in DMF which is comparable to those in typically used toluene. In DMSO even quantitative conversation was observed. Overall radiochemical yields of up to 40% and 60% in DMF and DMSO, respectively, were reached depending on the labelling yield of the first step. The fluorophenylpiperazine obtained was coupled in a third reaction step with 2-formyl-1H-indole-5-carbonitrile to yield the highly selective dopamine D4 ligand [18F]FAUC 316

Model and Knowledge Representation for the Reuse of Design Process Knowledge Supporting Design Automation in Mass Customization

Mass customization aims to meet individual requirements and, therefore, is one way to attract and retain customers—a key challenge in the design industry. The increase in design automation has offered new opportunities to design customized products at high speed in a way that is cost equivalent to mass production. Design automation is built upon the reuse of product and process knowledge. Ontologies have proven to be a feasible, highly aggregated knowledge representation in engineering design. While product and process knowledge from other lifecycle phases are represented in multiple approaches, the design process of the product as well as the adaption process of product variants is missing, causing breakpoints or additional iterations in design automation. Therefore, suitable knowledge representation tailored to design automation is still missing. Accordingly, this contribution proposes a novel knowledge representation approach to enable design automation for mass customization. Methodically, this novel approach uses semantic enrichment of CAD environments to automatically deduce information about a design task, design rationale, and design process represented by a formal ontology. The integration of the design process significantly differentiates the approach from previous ones. The feasibility of the approach is demonstrated by a bike crank customization process

Electrochemical generation of eletrophilic n.c.a. 18F-fluorinating reagents impossible?

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